The rapid developments in recombinant DNA methodology have allowed the production of polypeptides, proteins, and their analogs in unlimited quantities in a very short period of time. These developments have created a need to handle purification of these proteins from complex mixtures in highly efficient and predictable manners.
Recombinant DNA technology may be used for the production of desired polypeptides and proteins in host cells. Genes for desired proteins may be isolated from the genetic material of cells which contain the gene in nature or they may be chemically synthesized. The isolated or chemically synthesized gene may be inserted and expressed in host cell systems which produce protein products at high levels.
The desired protein products must then be isolated and recovered from the total amount of protein produced by the host cells. The purification of heterologous polypeptides produced by host cells can be very expensive and can cause denaturation of the protein product itself. An overview of protein purification techniques is provided in the Background Art section of U.S. Pat. No. 4,782,137 issued Nov. 1, 1988 to Hoppet al, which is incorporated herein by reference. Among the methods to purify proteins that are described in Hopp, the most commonly practiced are ion-exchange, hydrophobic chromatography, and gel filtration. The major disadvantage of these approaches are the lack of specificity of each technique. Thus, these techniques are unsuitable to achieve pure protein in high yields. Even small changes in amino acid composition may change the purification properties. A modified purification procedure needs to be developed and optimized for each new protein. In the ease of rDNA derived proteins, structural and functional consequences of heterologous gene expression (H. Bialy, Bio/technology, 5:884, 1987) are additional factors that may make it impossible to predict selection of these purification methods for a given protein.
Desired protein molecules may be isolated from complex mixtures by methods based on solubility differences. For example, isoelectric precipitation makes use of the alteration in protein solubility as a function of pH while fractionation with a solvent is based on the variation in protein solubility as a function of dielectric constant. Neutral salts, for example, ammonium sulfate, are employed to precipitate proteins due to decreased protein solubility based on high ionic strength of the salt. The drawback is that solvent fractionation can cause protein denaturation. Neither of these methods are capable of purifying proteins beyond a moderate level.
To avoid the negative elements of above techniques, affinity chromatography is often preferred. It is based on the ability of proteins to bind non-covalently but specifically with an immobilized ligand. When used alone, it can purify proteins from complex mixtures without significant loss. It requires the availability of the corresponding ligand for the desired protein; for example, an antibody for a protein antigen. It should be stressed that in some cases it may be difficult to obtain a specific ligand and such ligands do not exist for all proteins. As a result, this technique has not been applied as a universal method for protein purification.
To circumvent this limitation, recombinant DNA technology may be used to provide an affinity purification system where antibodies to a linker peptide may be used as an immunoaffinity ligand. This should provide a method that is capable of purifying recombinant proteins in one-step, using affinity chromatography, without sacrificing high yields. The Hopp patent relates to synthesis of a fusion peptide containing an antigenic linker peptide. The fusion peptide of Hopp is passed through a column containing immobilized antibodies which bind to the antigenic linker. Thus, the fusion protein may be isolated. The major drawbacks of this technique are that either the buffer conditions which are necessary to allow immunogenic complexing or the buffer conditions which must be present to terminate such complexes may denature the desired polypeptide product.
Immobilized Metal Ion Affinity Chromatography (IMAC) for fractionating proteins was first disclosed by Porath, I. et al., Nature 258:598-599 (1975). Porath disclosed derivatizing a resin with iminodiacetic acid (IDA) and chelating metal ions to the IDA-derivatized resin. Porath disclosed that proteins could be immobilized in a column which contained immobilized metal ions. The teachings of Porath include attaching a commonly used iminodiacetic acid (IDA) to a matrix followed by chelating a metal ion to the IDA-containing support resin. The proteins bind to the metal ion(s) through amino acid residues capable of donating electrons. Amino acids with potential electron donor groups are cysteine, histidines, and tryptophan. Proteins interact with metal ions through one or more of these amino acids with electron donating side chains. The actual mechanisms which result in binding of proteins to free metal ions or immobilized metal ions are not well known. A number of factors play a role; for example, conformation of the particular protein, number of available coordination sites on the immobilized metal ion, accessibility of protein side chains to the metal ion, number of available amino acids for coordination with the immobilized metal ion. Therefore, it is difficult to predict which protein will bind and with what affinity.
Smith et al. discloses in U.S. Pat. No. 4,569,794 that certain amino acids residues are responsible for the binding of the protein to the immobilized metal ions. However, if histidine side chains are involved in the binding the bound protein can be eluted by lowering the pH or using competitive counter ligands such as imidazole. Histidines containing di- or tripeptides in proteins have been used to show that IMAC is a specific and selective purification technique (U.S. Pat. No. 4,569,794). Accordingly, Smith et al. disclosed using recombinant DNA techniques to attach a metal chelating peptide to a desired polypeptide reproduced by recombinant techniques in order to provide a handle to the desired polypeptide. This handle can be used in protein purification by providing the chimeric protein with a metal chelating linker. Smith et al. disclosed several examples of metal chelating peptides, specifically those containing histidine, cysteine, methionine, glutamic acid, aspartic acid, lysine, and tyrosine. Smith et al. discloses that a fusion protein comprising a desired polypeptide with an attached metal chelating peptide handle may be purified from contaminants by passing the fusion protein and contaminants through columns containing immobilized metal ions. The fusion protein will chelate at the metal chelating peptide linker to the immobilized metal ions. The contaminants freely pass through the column and can thus be removed. By changing the conditions of the column, the chimerics can be released and then can be collected in pure form.
The present invention provides an improved method of purifying recombinant polypeptides and/or proteins. Recombinant polypeptides and proteins may be produced and purified by the method of the present invention with greater ease and therefore, more efficiently. Furthermore, these products may be recovered in pure, biologically active form. Using the method of the present invention, high yields of biologically active proteins may be efficiently recovered. The present invention also provides a method for removing the metal binding peptide from the purified chimeric protein without having to introduce a site-specific cleavage sequence.
The present invention is directed at the purification of biologically active recombinant polypeptides and/or proteins from bacterial or non-bacterial sources, most preferably those recombinant proteins expressed in a soluble form or secreted from the host as a fusion protein containing a metal chelating peptide. According to the present invention, the desired protein is first produced as a fusion protein which, in addition to the amino acid sequence of the desired protein, contains a linker peptide. The linker peptide of the present invention is a metal ion chelating peptide. Therefore, when the fusion protein according to the present invention is contacted with an immobilized metal ion containing resin, the fusion protein will be immobilized which will allow it to be separated from impurities.
It is possible to employ the commonly used IDA resin in IMAC for the purification of recombinant proteins having at least three alternating histidine residues. In a preferred embodiment of the invention, the amino acids that alternate with histidines are those which are specifically recognized by dipeptidylpeptidase I (DPP I). It is these discoveries that form the basis of this invention which is directed to fusion proteins comprising metal chelating affinity peptides, containing at least three alternate histidine residues, and a desired biologically active polypeptide or protein attached directly or indirectly to this/these metal chelating affinity peptides, a process for their synthesis by rDNA technology and a process for their purification by IMAC on commonly used IDA resins.
When the fusion protein is produced, the desired protein may be isolated and purified by passing the fusion protein through a column containing immobilized metal ions. The fusion protein chelates to the immobilized metal ions for a sufficient amount of time to allow it to be separated from other materials. The metal chelating peptides of the present invention provide superior and unexpected results over those taught or suggested by any of the prior art. The fusion protein of the present invention optionally contains a cleavage site that is located between the desired protein and the linker peptide and which is recognized by an endopeptidase. The purified fusion protein can then be cleaved at the scissile bond to separate the desired protein from the linker peptide. Alternatively, the metal chelating peptide according to the present invention may be removed from the desired protein portion by dipeptidylpeptidase I digestion.
The conditions needed in the purification step of the present method do not denature the fusion protein. The fusion protein may thus be purified to a biologically active final product in high yields using relatively few steps.